1. Field of the Invention
The present invention generally relates to the cultivation and growth of cells on laboratory, pilot plant, or industrial scales and, more particularly, to the cultivation and growth of cells in a plurality of individual microdroplets of liquid media which are interspersed within a matrix of hydrophobic microparticles.
2. Background Description
The culturing of microbial and animal or plant cells are crucial processes that are essential to the production of a wide array of useful chemical and biochemical products. Living cells are employed in such processes because they provide the essential elements necessary to economically synthesize many commercially valuable metabolic products.
Typically, growing cells are cultured either in liquid media (submerged cultivation) or on the surface of a solid nutrient(surface cultivation). Microorganisms such as bacteria and fungi can be cultured in either the surface or submerged method. Eukaryotic cells can be cultured in a submerged or suspended cell culture in rolling flasks or, where cell surface attachment is necessary, cells are grown to confluence in tissue culture flasks with liquid nutrient media placed above the cells. A suitable nutrient medium for microorganisms typically includes a carbon and energy source, an assimilable nitrogen source, oxygen (usually derived from surrounding air), and suitable pH conditions and additional factors which vary for a given microorganism, as one skilled in the art can readily appreciate.
With the surface method, nutrients are absorbed from contact with the media under the culture, oxygen is provided through contact with the air above the culture, and inhibitory metabolites seep down and away from the culture. Surface cultivation of microorganisms has the advantageous features of providing a plentiful oxygen source from the surrounding air and efficient removal of inhibitory metabolites through absorption from the surface medium. Also, contamination of surface culture can be relatively confined to a minimal surface area or a growing culture.
On the negative side, surface cultivation of microorganisms is not amenable to large scale production. The process of filling and inoculating numerous individual plates or dishes with culture and then individually harvesting each plate is extremely labor intensive. Furthermore, the storage of solid surface plates or dishes inoculated with microorganisms requires significant allocations of space in sophisticated incubators.
With the submerged method, a microorganism is cultured throughout the liquid media. Nutrients are absorbed from contact with the media surrounding the individual microorganisms, oxygen and are provided by various means of aeration that one skilled in the art can readily appreciate, and metabolites seep out and into the media. Usually, the nutrient media is also stirred continually, in order to evenly distribute the microorganisms.
The submerged cultivation process has the beneficial advantages of being less labor and space intensive than the surface method and can be used to produce large batches of cells in a relatively small space. The submerged method is thus the method of choice currently employed in most pilot and industrial scale production of cultured microrganisms and cells.
The submerged cultivation method does, however, require an extensive investment in equipment necessary for the large scale production of cell cultures. In addition, the end products that are the object of large scale submerged cultivation (i.e., the intracellular or extracellular metabolic products of cell and microbe growth) usually require further purification and concentration either from the liquid media or the cells therein. This additional isolation step is necessary because the concentration of product in the media is limited by the metabolites released into the media and the limited solubility of oxygen and/or other gases in the media.
Another major drawback to the large scale submerged cultivation method is the greater risk and effects of contamination that this method entails. In submerged cultivation, the complexity of the equipment necessary for bacterial fermentation systems, for example, provides more opportunities for a contamination event when compared to the surface growth methodology. Furthermore, once a culture is contaminated, the contamination quickly spreads throughout the agitated liquid media, resulting in the destruction of the entire batch of a culture.
It is therefore an object of the invention to provide for the growth of a microbe or cell culture with a hybrid method that both combines the beneficial features of submerged and surface cultivation while eliminating some of the negative features inherent in both procedures.
It is another object of the invention to provide an apparatus for the sterile growth of a cell culture in a collection of individual microdroplets. The invention provides for the growth of both prokaryotic and eukaryotic organisms. The invention is particularly suited to the aseptic cultivation of human, animal, plant or microbial cell populations.
It is further an object of the present invention to provide a method and apparatus for cell culture that substantially reduces the capital and labor costs of producing cell culture on a large scale.
It is further an object of the present invention to provide a method and apparatus for cell culture that substantially reduces the risks of contamination by opportunistic organisms.
It is further an object of the invention to provide a method and apparatus for cell culture that can be performed in a continuous flow or batch process.
It is further an object of the invention to provide a method for cell culture that eliminates the need to concentrate organisms following cell growth.
It is further an object of the present invention to provide a method and apparatus for cell culture that substantially increases the yield of products such as enzymes or proteins that are produced by the particular organisms cultured.
It is further an object of the present invention to provide a cell culture method that substantially increases the yield of products produced from genetically engineered cells such as, for example, vaccine products, biopesticides, antibiotics, and the like.
It is further an object of the invention to provide a method and apparatus for cell culture with substantially enhanced simplicity of operation and portability.
According to the invention, cells are cultivated in a plurality of individual microdroplets of liquid media. These microdroplets are created by aerosolizing liquid media that has been inoculated with the cells of interest and coating the aerosolized droplets with hydrophobic particles of solid material, such as silicon dioxide, for example. The individual microdroplets are stabilized within the hydrophobic solid particles, thereby providing a large number of small cell culture reactors. The coated microdroplets each provide a sterile environment for the individual microdroplets contained within the culture. Furthermore, the individual microdroplets each provide an optimum microenvironment with a reduced effect of potentially inhibitory metabolites and optimal accessability to aeration, resulting in substantial increases in the concentration of cells per liquid volume.